Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 568-574
ISSN: 2319-7706 Volume 3 Number 2 (2014) pp. 568-574
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Original Research Article
Isolation of indole acetic acid (IAA) producing rhizobacteria of Pseudomonas
fluorescens and Bacillus subtilis and enhance growth of onion (Allim cepa.L)
S.Reetha*, G.Bhuvaneswari, P.Thamizhiniyan and T.Ravi Mycin
Department of Botany, Annamalai University, Annamalai Nagar - 608 002, Tamilnadu, India
*Corresponding author
ABSTRACT
Keywords
Pseudomonas
fluorescens;
Bacillus
subtilis;
IAA;
PGPR
The present study was under taken for isolation of Pseudomonas fluorescens and
Bacillus subtilis from rhizosphere of Onion and analysis of these bacteria for
invitro indole acetic acid production and studying the effect of these bacteria on
plant growth of onion Plant. The soil samples were collected from rhizosphere of
onion plants from Botanical garden, Department of Botany, Annamalai University.
These two microorganisms are isolated by using KB and NB medium. Effect of
IAA producing Pseudomonas fluorescens and bacillus subtilis bacteria on plant
growth was studied by pot culture experiments by using sterilized air dried soil and
viable onion seeds . Both bacteria demonstrated increase in root length, shoot
length, root and shoot fresh and dry weight, on bacterial inoculated onion seeds
over control.
Introduction
The rhizosphere, representing the thin
layer of soil surrounding plant roots and
the soil occupied by the roots, supports
large active groups of bacteria (Villacieros
et al., 2003) known as plant growth
promoting
rhizobacteria
(PGPR)
(Kloepper et al.,1980). Plant growth
promoting rhizobacteria are known to
rapidly colonize the rhizosphere and
suppress soilborne pathogens at the root
surface (Rangajaran et al., 2003). These
organisms can also be beneficial to the
plant by stimulating growth (Bloemberg
and Lugtenberg, 2001; Moeinzadeh et al.,
568
2010).
Among
these
organisms,
Fluorescent Pseudomonads are considered
to be the most promising group of plant
growth promoting rhizobacteria involved
in biocontrol of plant diseases (Gardner et
al., 1984; Moeinzadeh et al., 2010). They
produce secondary metabolites such as
antibiotics
(Keel
et
al.,
1992),
phytohormones (Keel et al., 1992), volatile
compound Hydrogen cyanide (HCN)
(Defago and Haas 1990), and siderophores
(Neil and JB, 1995). Plant growthpromoting ability of these bacteria is
mainly because of the production of
Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 568-574
indole-3- acetic acid (IAA); (Patten and
Glick 2002), siderophores (Schippers et
al., 1987) and antibiotics (Colyer and
Mount1984).Rhizosphere is a rich niche of
microbes and should be explored or
obtaining
potential
plant
growth
promoting rhizobacteria (PGPR) which
can be useful in developing bio-inoculants
for enhancement of growth and yield of
crop plant. Bacteria predominates the
rhizosphere and take nutritional substances
(amino acid, vitamins and other nutrients)
released from plant tissues for growth
.The products of microbial metabolism
that are released into the soil also
influence the growth of plant. Interaction
between plant and microbes is well known
for beneficial effect and such free-living
soil bacteria isolated from the rhizosphere
of plants are known as plant growth
promoting rhizobacteria (Kloepper et al
1980). Some bacteria support plant growth
indirectly by producing antagonistic
substances or by inducing systemic
resistance against plant pathogen (Tilak et
al 2005).
indirectly by influencing bacterial 1aminocyclopropane-1carboxylic
acid
(ACC) deaminase activity (pedraza et al.,
2004) similarly role of phosphate
solubilising bacteria in plant growth and
development has been well documented
De.Freital et al ., 1997: Richardson et al.,
1999.
Materials and Methods
All glass ware first soaked in chromic acid
cleaning solution containing potassium
dichromate in 25% sulphuric acid or 3 hrs
and washed thoroughly in tap water. After
a second was in detergent solution. They
were again washed thoroughly in tap water
and finally rinsed in distilled water and
dried in oven.
Soil sampling
Soil sample were collected from the
rhizosphere of onion in the Botanical
garden Department of Botany Annamalai
University. Intact root system was dugout
and the rhizosphere soil samples were
collected in plastic bags.
Plant growth promoting rhizobacteria are
free living, soil-borne bacteria, which
enhance the growth of the plant either
directly or indirectly .The direct
mechanism involve nitrogen fixation.
Phosphorus
solubilisation,
HCN
production, production of phytohormone
such as auxin, cytokinin and gibberellins
and lowering of ethylene concentration
(Kloepper et al., 1989; Glick, 1995; Glick
et al., 1999).In wheat Azospirillum
brasilense of wheat seedlings increase the
number and length of lateral roots
(Barbieri et al., 1986). Importance of IAA
production by PGPR has been widely
acknowledged (Kennady et al., 2004;
Rosech et al., 2007 Ashrafuzzaman et al.,
2009). IAA selected by stimulating plant
cell elongation or cell division or
Isolation of bacteria from rhizosphere
soils
The plant growth promoting rhizobacteria
were isolated from the rhizosphere soil
sample by serial dilution plate methods.
Appropriate dilution was spread on NB
and KB agar plates. Plates were incubated
at 28±2 c for 24 to 48 hrs. Colonies were
picked from these plates and maintained as
pure culture in respective media with
periodic transfer to fresh media and
stocked for further use
Production of indole acetic acid
The selected antagonistic bacterial strains
were grown in 5ml of nutrient broth
569
Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 568-574
medium in test tube or 24 hrs. After
incubation period, bacterial culture were
harvested and centrifuged at 100000xg
for 15min at 4 c. Two drops of
orthophosphoric acid were added to 2ml of
cell free supernatant and the development
of colour was observed the presence of a
pink colour indicate positive reaction for
indole acetic acid and yellow colour
indicate negative reaction.
Results and Discussion
In the present studies were carried out to
isolate the bacterial culture from the
rhizosphere of onion plant. The isolates of
Psedomonas fluorescens and Bacillus
subtilis were screened for their plant
growth promoting activities of Indole
acetic acid (IAA) production from the
rhizosphere of onion plant.
Method of seed treatments
Indole acetic acid production (IAA)
(µg/ml)
Bio control agents were grown in
respective medium on rotating shaker
(150rpm) for 2 days and centrifuged at
10,000 rpm for 5min the pellet was mixed
with sterile carboxyl methyl cellulose
(CMC) suspension .Onion seeds surface
sterilized with sodium hypochlorite
solution were placed in CMC cell
suspension and air dried inside laminar air
flow chamber (jagadeesh, 2000).The bio
coated seeds were transplanted into the
field .
The results originated from both
qualitative and quantitative assays of IAA
reflected the ability of two tested
microorganisms to produce indole
compounds.
The
two
tested
microorganisms exhibited a pink to red
colour with a little variation in intensity. In
the quantitative measurements, the highest
value of IAA production was obtained by
P. fluorescens followed by B. subtilis, as
they
produced
(15.38±0.537)
and
(12.67±0.325) respectively.
Shoot length and root length (cm)
Root length (cm)
Three plants were randomly selected for
recording the root length and shoot length
of onion plants. They were measured by
using centimeter scale.
Data presented in Table 2 showed that the
effect of different biocontrol applications
on the root length of Onion. The highest
root length was recorded in Pseudomonas
fluorescens (7.500 ± 0.100) treated plants
when compared to Bacillus subtilis (7.333
± 0.152) and control (5.666 ± 0.404).
Fresh and dry weight (mg/g dry wt.)
Three plant samples were randomly
selected at experimental pot. They were
separated into root and shoot. Their fresh
weight was taken by using an electrical
single pan balance. The fresh plant
materials were kept in a hot air oven at
80 C for 24 hr and then their dry weight
were also determined
Shoot length (cm)
Significantly higher shoot length was
produced in Pseudomonas fluorescens
(27.41 ± 0.102) compared to Bacillus
subtilis (25.30 ± 0.173) and control (25.10
± 0.34).
570
Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 568-574
multiple plant growth promoting activity.
Compare to these two bacteria,
Pseudomonas fluorescence have potential
to produce high amount of IAA production
to Bacillus subtilis and control. The result
shows that these isolates possessed PGP
activities the range of percentage of IAA
production of these bacterial activities are
varied greatly. Mahalakshmi and Reetha
(2009), (Saitou N and Nei M 1987)
reported similar results in the assessment
of PGP activities of bacterial isolates from
the rhizosphere of tomato. The rhizosphere
microorganism especially fluorescent
pseudomonas, have exceptional ability to
promote the growth of host plant by
various mechanism to suppress plant
diseases including production of powerful
sideraphores (O sullivan and O Gara
1992): Has and Defago 2005).
Shoot Fresh weight of plant (g)
The data pertaining to the effect of
biocontrol agents on shoot fresh weight of
onion plants are depicted in Table 1. The
fresh shoot weight of onion plants was
significantly high in Pseudomonas
Fluorescens (3.590 ± 3.777), compare to
Bscillus subtilis (2.830 ± 0.060). The least
weight of whole plant was observed in the
uninoculated control (2.556 ± 0.297).
Root Fresh weight of plant (g)
The data pertaining to the effect of
biocontrol agents on Root fresh weight of
onion plants are depicted in (Table 1). The
fresh root weight of onion plants was
significantly high in Pseudomonas
fluorescens (0.953 ± 0.047), compare to
Bacillus subtilis (0.556 ± 0.030). The least
weight of whole plant was observed in the
uninoculated control (0.380 ± 0.057).
The property of synthesizing indole acetic
acid is considered as an effective tool for
screening beneficial microorganism as
there have been reports suggesting that
IAA producing bacteria having profound
effect on plant growth(Kuklinsky et al
1999),Ali B and Hashin S 2007) The
improved plant growth by PGPR is due to
its ability to produce phytohormones such
as indole acetic acid (IAA) (Pattern and
Glick, 2002), gibberellic acid (Mahmoud
et al. 1984), cytokinin (Tien et al., 1979)
and its ability to produce ACC-deaminase
to reduce the ethylene in the roots of the
developing plants thereby increasing the
root length and growth (Penrose and
Glick, 2001). B. subtilis isolates improved
the germination, emergence; increased
root growth and nodulation in groundnut
along with consistent colonization were
reported by Turner and Backman (1991).
Tsavkelova et al. (2006) stated that
Bacillus involves the modulation of plant
development through the production of
phytohormones. Thus, several Bacillus
Shoot Dry weight of plant (g)
The results of the dry weight of shoot
onion under two different biological
control on recorded in (Table 2). Shoot dry
weight of plant was highest in the
Pseudomonas fluorescens (0.260 ± 0.100),
and Bacillus. Subtilis (0.233 ± 0.005).The
least dry weight was seen in the control
plants (0.226 ± 0.025).
Root dry weight of plant (g)
The results of the Root dry weight of
onion under two different biological
control on recorded in (Table 2). Root dry
weight of plant was highest in the
Pseudomonas fluorescens (0.600 ± 0.010),
and Bacillus Subtilis (0.500 ± 0.010).The
least dry weight was seen in the control
plants (0.433 ± 0.057).
Pseudomonas and Bacillus bacteria posses
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Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 568-574
Table.1 Effect of PGPR inoculation on Growth parameters of onion plant
Treatment
Shoot length
Root length
Shoot fresh weight Root fresh weight
25.10 ± 0.34
5.666 ± 0.404
2.556 ± 0.297
0.380 ± 0.057
Pseudomonas fluorescence 27.41 ± 0.102
7.500 ± 0.100
3.590 ± 3.777
0.953 ± 0.047
25.30 ± 0.173
7.333 ± 0.152
2.830 ± 0.060
0.556 ± 0.030
Control
Bacillus subtilis
The values are mean ± SD for three samples in each group
Table.2 Evaluation of the ability of the microorganisms to exhibit
IAA Production in in-vitro condition
Treatment
Shoot dry weight
Root dry weight
Control
0.226 ± 0.025
0.433 ± 0.057
Pseudomonas fluorescence
0.260 ± 0.100
0.600 ± 0.010
Bacillus subtilis
0.233 ± 0.005
0.500 ± 0.010
IAA Production
Colour intensity
µg/ml
+++
++
15.38±0.537
12.67±0.325
The values are mean ± SD for three samples in each group
species are capable of producing auxin
that might stimulate root proliferation and
nutrient uptake (Spaepen et al., 2007).
Khan et al. (2003) described that soil
application of Aspergillus awamori or P.
fluorescens increased the dry weight of
shoot by 19.0% and 9.5% respectively,
compared to uninoculated control.
Combined application of a P. fluorescens
along with Bradyrhizobium in groundnut
significantly enhanced groundnut root and
shoot dry weight, nodule number, nodule
dry weight, and per cent nitrogen content
of shoot (Vikram et al., 2007). In the
quantitative measurements, the highest
value of auxin production was obtained P.
fluorescens followed by B. subtilis and T.
harzianum. This is in consonance with the
work of (and Verma et al. (2010) who
found that high proportion of rhizomicroorganisms are able to produce plant
growth hormone, i.e., indole acetic acid,
which acts to stimulate plant growth and
provides it with more branching and larger
surface area. Thus these IAA producing
bacteria like pseudomonas fluorescens and
bacillus subtilis were further studied or
their effect on plant growth under
controlled conditions. Data obtained from
the pot experiments demonstrated positive
effect on root elongation of treated plants
over the control .This indicate both
bacteria have the efficiency to improve the
root and shoot length of development of
the plant and these can be considered as
plant growth promoters.
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